Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk

Abstract

During the development of the peripheral nervous system there is extensive apoptosis, and these neuronal corpses need to be cleared to prevent an inflammatory response. Recently, Jedi-1 and MEGF10, both expressed in glial precursor cells, were identified in mouse as having an essential role in this phagocytosis (Wu et al., 2009); however, the mechanisms by which they promote engulfment remained unknown. Both Jedi-1 and MEGF10 are homologous to the Drosophila melanogaster receptor Draper, which mediates engulfment through activation of the tyrosine kinase Shark. Here, we identify Syk, the mammalian homolog of Shark, as a signal transducer for both Jedi-1 and MEGF10. Syk interacted with each receptor independently through the immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains. The interaction was enhanced by phosphorylation of the tyrosines in the ITAMs by Src family kinases (SFKs). Jedi association with Syk and activation of the kinase was also induced by exposure to dead cells. Expression of either Jedi-1 or MEGF10 in HeLa cells facilitated engulfment of carboxylated microspheres to a similar extent, and there was no additive effect when they were coexpressed. Mutation of the ITAM tyrosines of Jedi-1 and MEGF10 prevented engulfment. The SFK inhibitor PP2 or a selective Syk inhibitor (BAY 61-3606) also blocked engulfment. Similarly, in cocultures of glial precursors and dying sensory neurons from embryonic mice, addition of PP2 or knock down of endogenous Syk decreased the phagocytosis of apoptotic neurons. These results indicate that both Jedi-1 and MEGF10 can mediate phagocytosis independently through the recruitment of Syk.

Figure 1.

Jedi-1 and MEGF10 contain ITAMs required for Syk interaction. A, The intracellular domains of Draper, Jedi-1, and MEGF10 were aligned by NCBI Cobalt Alignment. ITAM motifs are underlined. B, Syk-myc or Zap-70-myc were transfected into HEK293 cells stably expressing Jedi-GFP. Jedi-GFP was immunoprecipitated with anti-GFP, and anti-myc was used to detect Syk-myc and Zap-70-myc. C, Syk-myc was transfected into HEK293 cells stably expressing WT or mutated Jedi-GFP. Anti-GFP was used to immunoprecipitate WT and mutant Jedi-GFP. D, Syk-myc and WT or mutated MEGF10-GFP were transiently transfected in HeLa cells. WT and mutant MEGF10-GFP were immunoprecipitated with anti-GFP.

Figure 2.

Jedi-1 ITAM tyrosines are required for engulfment. Jedi-GFP, mutants of Jedi-GFP or LRP together with GFP was transfected into HeLa cells and microspheres were added for 2 h, then the cells were rinsed and fixed. A, Confocal images are shown with Jedi or GFP-expressing cells (green) containing engulfed microspheres (red). Cell nuclei are shown in blue. B, The expression of WT and mutant Jedi-GFP were confirmed by Western blot. C, The percentage of HeLa cells, transfected with WT or mutant Jedi or LRP, engulfing at least one microsphere was quantified (p = 0.0016 for LRP, p = 0.00009 for Jedi, n = 3–6). D, GFP, WT Jedi-GFP, or mutant Jedi-GFP was transfected into glial cells in cocultures of E13.5 DRG neurons and glia. Neuronal death was induced by NGF withdrawal, and the percentage of transfected glia engulfing at least one apoptotic body was quantified by confocal analysis (p = 0.048, n = 3).

Figure 3.

MEGF10-mediated engulfment requires its ITAM domain and is not additive with Jedi. A, Jedi and MEGF10 were transfected alone or together, or mutant MEGF10 was transfected, into HeLa cells. Expression levels were analyzed by Western blot. B, Microspheres were added, and the percentage of transfected cells engulfing at least one microsphere was quantified by confocal microscopy (MEGF10, p = 0.0003; MEGF10 + pcDNA3, p = 0.0034; Jedi+pcDNA3, p = 0.0045; Jedi+MEGF10, p = 0.0005, n = 3).

Figure 4.

Syk associates with Jedi-1 and becomes active upon stimulation with apoptotic CHO cells. HEK293 cells stably expressing Jedi-GFP were transfected with Syk-myc and 24 h later, apoptotic CHO cells were added for the indicated times. Coimmunoprecipitation of Syk-myc with Jedi-GFP was detected with anti-myc. Phospho-Syk was detected in the lysates by Western blotting with a phospho-Syk-specific antibody.

Figure 5.

Syk and SFKs are required for Jedi-1 or MEGF10-mediated engulfment. A and B, HeLa cells were transfected with GFP, Jedi-GFP, or MEGF10-GFP, and after 48 h, 1 μm Syk inhibitor (BAY 61-3606) or 1 μm PP2 was added to the HeLa cells 1 h before addition of microspheres for 2 h. Cells were rinsed, fixed, and engulfment of microspheres was detected by confocal microscopy and quantified. The Syk inhibitor significantly reduced Jedi-mediated (p = 0.015, n = 5) and MEGF10-mediated (p = 0.026, n = 3) engulfment. Similarly, PP2 significantly decreased the engulfment mediated by Jedi (p = 0.016, n = 5) or MEGF10 (p = 0.024, n = 3).

Figure 6.

Altered Syk expression or inhibition affects the engulfment of neurons by glial precursors. A, Syk-myc, GFP, and scrambled or Syk shRNA were transfected into HEK293 cells. Lysates were immunoblotted with anti-myc and anti-GFP. B, Confocal images depicting cocultures of E13.5 DRG neurons and glial precursor cells transfected with GFP and scrambled or Syk shRNA, GFP or Syk-myc. Neuronal death was induced by removing NGF for 48 h. Transfected glia are depicted in green and nuclei in red. C, Scrambled or Syk shRNA, and GFP were transfected into glia in cocultures of DRG neurons and glia and the percentage of transfected glia that were engulfing apoptotic neurons was quantified (Syk shRNA, p = 0.021; Syk over expression, p = 0.022; n = 4). D, Cocultures were transfected with GFP, Jedi-GFP, or MEGF10-GFP, and a Syk inhibitor (BAY 61–3606) was added for the last 24 h. Engulfment of apoptotic bodies was quantified by confocal microscopy. Expression of GFP, Jedi (p = 0.022, n = 3) or MEGF10 (p = 0.0056, n = 3) significantly increased engulfment, but this was prevented by treatment with the Syk inhibitor (Jedi, p = 0.0022; MEGF10, p = 0.025, n = 3).

Figure 7.

SFKs phosphorylate Jedi-1 and MEGF10 at ITAM tyrosines, increasing Syk binding. A, HEK293 cells stably expressing Jedi-GFP were untransfected or transfected with Syk-myc. Jedi-GFP immunoprecipitates were immunoblotted with anti-phosphoTyrosine, anti-GFP and anti-myc. B, HEK293 cells were transfected with Jedi-GFP or MEGF10-GFP and Lck, Lyn, Src, or Yes. Jedi-GFP or MEGF10-GFP were immunoprecipitated with anti-GFP and immunoblotted with anti-phophotyrosine. (The images for each kinase and its control are from the same blot, but not always from adjacent lanes. The lanes were put next to each other for the figure). C, D, HEK293 cells expressing WT or mutant Jedi-GFP, or WT or mutant MEGF10-GFP, were transfected with Fyn. Jedi-GFP or MEGF10-GFP was immunoprecipitated and the precipitates were immunoblotted with anti-phosphoTyrosine and anti-GFP. E, F, HEK293 cells stably expressing Jedi-GFP or MEGF10-GFP were transfected with Syk-myc and Fyn, Lck, Lyn, Src, or Yes. Jedi-GFP or MEGF10-GFP was immunoprecipitated with anti-GFP and the precipitates were immunoblotted with anti-phosphoTyrosine, anti-GFP and anti-myc.

Figure 8.

Jedi-1 and MEGF10 independently signal engulfment via SFKs and Syk. When Jedi-1 and MEGF10 are stimulated by apoptotic cells, the SFKs Fyn, Lck, Lyn, and Yes phosphorylate the ITAM tyrosines of Jedi-1, and Fyn, Lck, and Lyn phosphorylate the ITAM tyrosines of MEGF10, resulting in increased Syk interaction. Src can phosphorylate the ITAM tyrosines of Jedi-1 and MEGF10, but it does not increase Syk binding. The stoichiometry of Syk binding to the receptors is not known, but mutation of a single ITAM tyrosine markedly reduced the interaction. Syk association with Jedi-1 and MEGF10 leads to engulfment of apoptotic neurons by glial cells.